Our group has developed a spectroscopic method allowing measurement of the transverse location (depth) of sites in a membrane at the angstrom level of resolution. It involves fluorescence quenching measurements using phospholipids carrying nitroxide quenchers at different depths. Simple algebraic expressions are then used to calculate depth. Procedures have now been developed which allow accurate measurements over wide range of depths. In the next project period, the method will be used to investigate a fundamental issue of membrane structure, namely, the rules governing the relationship of the chemical structure of membrane-interacting molecules to their depth. As part of this aim depth will be measured for s systematic series of hydrophobic and lipid-linked probes we will prepare such that the attachment site, probe polarity, and the nature of the reactive group on the probe is varied. The effect of cholesterol on depth will also be determined. This is of particular interest in view of recent studies showing that cholesterol and sphingolipid-rich domains may exist in cellular membranes. In collaborative studies, the regulation of the formation of such domains may exit in cellular membranes. In collaborative studies, the regulation of the formation of such domains by lipid composition, and their interactions with proteins will be examined. The quenching method will also be used to determine how amino acid composition regulates the structure of membrane inserted polypeptides. This will be examined by depth measurements on derivatives of known transmembrane inserted polypetides. This will be examined by depth measurements on derivatives of known transmembrane a-helical hydrophobic peptides into which different types and numbers of polar residues have been introduced at specific positions. Measurement of the depth of a single Trp residue in these peptides, in conjunction with circular dichroism and energy transfer measurements, will allow facile analysis of the effects of the substitutions on peptide structure, and the formulation of rules relating transmembrance orientation to sequence. The method will also be applied to the high resolution topography of membrane proteins. This will be done by measuring the depth of single fluorescent groups introduce at a series of residues by site-directed mutagenesis. First, the calibration of the method and identification of the best fluorescent labels for such studies will be completed by a fuller comparison of the theoretical and experimental depths for single site-labeled transmembrane peptides. Then, the method will be applied to a membrane protein for which a series of single Cys mutants are now available, the a-hemolysin of S. Aureus.